JPS6112894B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to hepatitis B, and more particularly to hepatitis B antigen and a method for purifying hepatitis B antigen for use as a vaccine. Hepatitis B is one of two types of viral hepatitis and is primarily a pathological change that occurs in the liver, resulting in systemic infection. The disease primarily affects adults and is largely maintained by transmission of infection from long-term carriers of the virus. The usual methods of spread are through saliva, venereal contact, or exposure to contaminated needles and needles, unsterilized dental instruments, and spray-injected infected blood through the skin broken by blood cuts or scratches. The incubation period for type B hepatitis is relatively long, ie, 6 weeks to 6 months elapses between infection and the beginning of clinical symptoms. The illness usually begins with fatigue and lack of appetite, often accompanied by muscle pain and abdominal discomfort. Later, jaundice, black urine, white stools, and weak hepatomegaly appear. In some cases, the onset is rapid with early appearance of jaundice associated with fever, chills, and leukocytosis. In other cases, jaundice is not detected and the patient only perceives a "flu-like" illness. It is estimated that the majority of hepatitis infections result in mild anjaundice. Although qualitatively similar to viral hepatitis A, the disease may be caused by blood or other clinical specimens (saliva, urine,
Australian antigen particles in bile, stool) [currently
It is easily diagnosed by the appearance of HBs Ag (surface antigen). A relatively large population of spherical particles (10 ¹⁴ -10 ¹⁵ /ml) occurs in the blood of infected individuals. The particles are 18-22 nm in diameter and have antigenic determinants similar to the surface of the 42 nm Dane particle, the virus of hepatitis B (now called HBV). US Pat. No. 3,735,004 describes how boiled infectious serum is used to vaccinate children.
It has been shown that such sera produced antibodies against HBsAg (anti-HBs) and provided protection against challenge. The dose of boiled serum used was approximately 1×
This corresponds to 10 ¹² HBs particles. Recipients of such serum did not develop clinical or biochemical disease. However, such serum is not suitable for use as a vaccine due to its impure nature, lack of reproducibility and non-quantification. It is therefore an object of the present invention to provide a vaccine against hepatitis B. The second objective is to purify the hepatitis B antigen by removing foreign and undesirable proteins and/or antigens. A further object is to provide a method for removing foreign and undesirable proteins or antigens from hepatitis B antigens. Still another object is to provide a pharmaceutical preparation for administering purified hepatitis B antigen as a vaccine. These and other objects of the invention will become apparent from the following description. A partially purified concentrate from clarified plasma containing hepatitis B antigen is subjected to the following operating sequence. (1) The concentrate is treated with pepsin at a pH in the range in which pepsin is active. (2) The concentrate is treated with urea. and (3) if necessary, the concentrate is treated with formaldehyde. The resulting highly purified, reproducibly characterized hepatitis B antigen, separated from foreign and undesirable proteins and/or antigens, is useful as a vaccine. The starting material for the purified hepatitis B surface antigen (HBsAg) of the present invention is plasma obtained from a donor, for example by plasmaphoresis. The level of antigen is measured by known methods such as radioimmunoassay, passive hemagglutinin reaction or complement fixation reaction. The plasma is cooled and the cold precipitate that forms is removed by light centrifugation. Residual clarified plasma containing HBsAg is removed by one or more techniques such as isopycnic banding, rate zonal banding, or precipitation with, for example, polyethylene glycol, ammonium sulfate, sodium sulfate, etc. Concentrated. This partially purified concentrate is the starting material for the present invention. In the same density zone, the partially purified concentrate is contacted with a liquid medium having a density gradient encompassing the required specific antigen density. The liquid medium is then subjected to ultracentrifugation in order to obtain an equilibrium distribution of serum components by means of a density gradient according to their individual densities. Successive fractions of the medium are then displaced and fractions containing the desired antigen are separated. The application of this technique to the purification of Australian antigens is described in German specification No. 2049515 and US Pat. No. 3,636,191. In rate zonal banding, a partially purified concentrate is subjected to ultracentrifugation in contact with a liquid medium with a density gradient, where the rate zonal technique is used at such a rate and duration that equilibrium is not achieved. Ru. HBsAg and other residual serum components are then distributed in the medium according to the sedimentation coefficient in the medium. The liquid medium used in the treatment method may have any density gradient within a suitable range. Suitable solutes for such solutions include, for example, sucrose, potassium bromide, cesium chloride, sodium bromide, potassium tartrate, and the like. The density zone stage is usually separated using a centrifuge, e.g. Electronucleonics-K.
(Electronucleonics-K) by filling a stationary rotor with saline and then displacing the saline upward with aliquots of liquid medium solution of increasing density until a step gradient is formed. It can be done. Plasma is introduced from the bottom into the head of the rotor displacing the densest solution. Centrifugation is performed in a controlled system where the centrifugation speed is programmed to prevent mixing during the initial reorientation phase.
When equilibrium is reached and the product reaches its characteristic density,
The rotor is decelerated, and by controlling the speed using the same system as described above, it is possible to prevent mixing when reorienting to the original state. The gradient is then ejected from below and the characteristic density cross sections are collected. Similar techniques are used in rate zonal banding. The characteristic density cross section from this band is a partially purified concentrate of hepatitis B antigen. The protein concentration of the partially purified concentrate is then adjusted to about 40-200 micrograms per ml with the addition of sterile pyrogen-free phosphate buffered saline (PBS).
The solution is then adjusted to a pH of about 2-4, preferably about 25 with HCl.
acidified at °C. An aqueous solution of purified pepsin, preferably prepared from crystalline pepsin, is added to contain about 1 microgram of pepsin per about 40-500 micrograms of protein. The solution is incubated for about 8 to 24 hours, typically at a temperature of about 30-38°C, until digestion of foreign proteins by pepsin is complete. The solution is then brought to a pH of approximately 7 by addition of a base such as NaOH. The pepsin-digested material is concentrated by ultrafiltration at about 5°C and the urea is added until the final concentration is effective to dissociate the proteinaceous material, typically about 4M to 8M (preferably a highly purified crystalline grade). ) is added. The mixture is then held at an elevated temperature, typically about 30-38° C., for about 16-24 hours for further purification. The reaction mixture is clarified by filtration and chromatographed on a column to remove urea, pepsin, and pepsin digestion residues. The column is filled with a sterile, pyrogen-free, physiologically acceptable buffer that is compatible with the column, e.g. PBS, NaH ₂ PO ₄ and
It is eluted with a mixture of Na ₂ PO ₄ or three. Known methods such as radioimmunoassay,
Fractions identified as containing HBsAg by complement fixation and UV spectroscopy are pooled in sterile, pyrogen-free, physiologically acceptable buffers (some examples of which are listed above).
diluted to a level of approximately 20 micrograms HBsAg per ml. The diluted pooled fractions are sterile filtered. If necessary, formaldehyde is added to the filtered batch to inactivate virus at a known concentration, eg, about 50 to 200 micrograms per ml. The mixture is then heated to about 50-100℃ at about 30-38℃.
Incubated for hours. A sterile solution of a physiologically acceptable neutralizing agent, such as NaHSO ₄ , is added to substantially neutralize the formaldehyde. The materials are aseptically dispensed into glass vials and stored for future use. The purified hepatitis B surface antigen of the present invention is a highly purified and reproducible product, with a particle size of approximately 18-22 nm in diameter, E
¹ % ₂₇₈ ohm is typically about 52-54 ohm and gives properties with a UV spectrum as shown in the figure. Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples unless it exceeds the gist thereof. Example 1 The rotor of a centrifuge Electronureonics K was filled with 8400 ml of phosphate buffer. After the rotor was rotated to 10,000 rpm to degas the system, the following stepwise gradients, in descending order of density, were pumped into the bottom of the stationary rotor. 1 10% NaBr 2400ml ρ = 1.08 2 20% NaBr 1000ml ρ = 1.17 3 30% NaBr 1500ml ρ = 1.28 4 40% NaBr 3500ml ρ = 1.41 1750ml of plasma containing Australian antigen was replaced with 1750ml of 40% NaBr from the bottom of the rotor. while pumping into the head of the stationary rotor. The rotor was accelerated to 30,000 rpm and the test was run at this speed for 4 hours. Next, the rotor was stopped, and 1750 ml of 40% NaBr was injected into the bottom of the rotor while pushing plasma toward the head. An additional 1750 ml of fresh plasma containing Australian antigen was injected into the head of the rotor with an equal volume of 40% NaBr displacing from the bottom of the rotor.
The rotor was then rotated at 30,000 rpm for 18 hours. After stopping the rotor, the density range 1.21~1.24
1000 ml of HBsAg-rich material was collected and dialyzed against degassed phosphate buffer. The rotor was then filled with the degassed phosphate buffer described above and the step gradient described below was injected into the bottom of the stationary rotor. 1 5% sucrose 2400ml ρ = 1.02 2 15% sucrose 1750ml ρ = 1.06 3 25% sucrose 1750ml ρ = 1.10 4 50% sucrose 2500ml ρ = 1.23 HBsAg-rich substance from the zonal stage of NaBr isodensity
1000 ml was injected from the bottom of the rotor into the head of the rotor, replacing 1000 ml of 50% sucrose. The rotor was then rotated at 28000 rpm for 18 hours. After stopping the rotor, the HBsAg-rich material in the density range 1.135-1.165 was collected. This product was then diluted to 40 micrograms protein per milliliter to yield 56 liters of material. The diluent is phosphate buffered saline. A 56 liter batch (40 μg/ml) of hepatitis B antigen (HBsAg) partially purified by zonal centrifugation was acidified to PH2 by addition of IN hydrochloric acid with stirring at room temperature. 56 ml of a 1 mg/ml crystalline pepsin solution in distilled water was added. The final concentration of pepsin was 1 μg/ml. HBsAg batch at 37℃
Incubate for 16 hours and adjust the pH by adding 1N sodium hydroxide.
7 was added. The pepsin digested batches were concentrated 295-fold by ultrafiltration using an Amicon device equipped with an XM100A membrane. Pepsin-degraded non-antigenic proteins passed through the membrane into the ultrafiltrate, but HBsAg particles were retained. Solid urea was added to the concentrate to produce a 4-8 molar urea solution and then incubated at 37°C for an additional 16 hours. The urea-treated HBsAg concentrate was clarified by filtration through a fiberglass filter and chromatographed on Sephadex G-150. The antigen was eluted in the void volume, liberating urea, pepsin, and other protein impurities. The fraction containing purified HBsAg was diluted to working levels and sterile filtered. formalin 36
C. for 72 hours at a concentration of 90-100 .mu.g/ml and further ensured against the possible presence of infectious viruses. At the end of formalin treatment, excess formaldehyde was neutralized with sodium bisulfite. The purified product has an E ₁ % of 23.8% relative to the zonal centrifugation starting material.
has an E ₁ % of 52.3 compared to . The purified product consists of spherical particles with a diameter of 18-22 nm and a UV spectrum as shown in the accompanying drawings. A summary of the purification, biophysical properties is shown in the following table.

Table: Example 2 Four out of a group of six chimpanzees developed antibodies when injected subcutaneously with a single dose (1.0 ml) containing 20 micrograms of the hepatitis B surface antigen of the invention. After two allogeneic booster doses 4 weeks apart, 5 of 6 chimpanzees developed antibodies. Example 3 A test regarding the infectivity of the hepatitis B surface antigen of the present invention was conducted as follows. Sixteen chimpanzees were divided into three groups. Group A (6 chimpanzees) has BOB hepatitis B virus 1.0
ml was inoculated intravenously. Group B (4 chimpanzees)
were intravenously inoculated with 1.0 ml of the hepatitis B surface antigen of the present invention. Group C (6 chimpanzees) was a control group and was not vaccinated. All chimpanzees in Group A had an obvious clinical hepatitis B infection (either antiignemia, enzyme elevation and/or antibody response) within 40 weeks. None of the chimpanzees in Groups B or C exhibited overt clinical hepatitis B infection over a similar period of 40 weeks. Example 4 Three grivet monkeys (6 monkeys in each group) were injected subcutaneously three times at 4 week intervals with 1.0 ml doses containing a variable of the antigen of the invention. The following table shows the dose received in each group at each injection and the number of animals in total, of which groups either showed antibody formation before the next injection or the last injection within 4 weeks. This is what we do.

Importantly, more than 50% of the animals showed an antibody response after three doses of vaccine at the 2 microgram dose level. Example 5 Three groups of guinea pigs (each group
(14 mice) were injected subcutaneously at 0.14 and 56 day intervals with a volume of 1.0 ml containing variables of the antigen of the invention. The following table shows the doses of each injection administered to each group and the number of animals in total, among which the groups show the antibody formation when tested on days 0, 28, 56 and 84.

Importantly, more than 80% of the guinea pigs developed antibodies after three doses of vaccine at the 0.5 microgram dose level.

[Brief explanation of the drawing]

The figure shows the ultraviolet spectrum of the hepatitis B antigen of the present invention.

Claims (1)

[Claims] 1. Treating the concentrate with an amount of pepsin effective to digest proteinaceous substances within the pH range at which pepsin is enzymatically active, and an amount of urea effective to dissociate proteinaceous substances. containing hepatitis B antigen, characterized by treating the concentrate with a hepatitis B antigen to remove pepsin, pepsin degradation products and urea, and if necessary adding formaldehyde to a concentration effective to inactivate the virus. A method for purifying hepatitis B antigen from a partially purified concentrate obtained from purified plasma. 2. The method of claim 1, wherein the pH of the concentrate is adjusted to about 2 with HCl. 3. The method of claim 1, wherein pepsin is added to a concentration of about 1 microgram/ml for every about 40 to 500 micrograms of protein. 4. The method of claim 1, wherein urea is added to a concentration of about 4-8M. 5 Add almaldehyde, then pepsin,
The method of claim 1 for removing pepsin degradation products and urea. 6. The method of claim 5, wherein formaldehyde is added to a concentration of about 50 to 200 micrograms/ml. 7. The method of claim 5, wherein the removal is carried out by filtration and chromatography. 8 Particle size diameter approx. 18-22nm, E ¹ % _278o m approx. 5
2
~54 and having a UV spectrum substantially as shown in the figure. 9 E ¹ % _{278 o} m approx. 52
Antigen of the nuchal.